Mutagenesis is a powerful tool in the study of protein structure and function. Mutations can be made in the nucleotide sequence of a cloned gene encoding a protein of interest and the modified gene can be expressed to produce mutants of the protein. By comparing the properties of a wild-type protein and the mutants generated, it is often possible to identify individual amino acids or domains of amino acids that are essential for the structural integrity and/or biochemical function of the protein, such as its binding and/or catalytic activity. The number of mutants that can be generated from a single protein, however, renders it difficult to select mutants that will be informative or have a desired property, even if the selected mutants that encompass the mutations are solely in putatively important regions of a protein (e.g., regions that make up an active site of a protein). For example, the substitution, deletion, or insertion of a particular amino acid may have a local or global effect on the protein.
Previous methods for mutagenizing polypeptides have been either too restrictive, too inclusive, or limited to knocking out protein function rather than to gaining or improving function. For example, a highly restrictive approach is selective or site-directed mutagenesis which is used to identify the presence of a particular functional site or understand the consequences of making a very specified alteration within the functional site. A common application of site directed mutagenesis is in the study of phosphoproteins where an amino acid residue, that would ordinarily be phosphorylated and allow the polypeptide to carry out its function, is altered to confirm the link between phosphorylation and functional activity. This approach is very specific for the polypeptide and residue being studied.
Conversely, a highly inclusive approach is saturation or random mutagenesis that is designed to produce a large number of mutations encompassing all possible alterations within a defined region of a gene or protein. This is based on the principle that, by generating essentially all possible variants of a relevant protein domain, the proper arrangement of amino acids is likely to be produced as one of the randomly generated mutants. However, in practice, the vast number of random combinations of mutations generated can prevent the capacity to meaningfully select a desired candidate because of the presence of the so-called “noise” of so many undesired candidates.
Another approach, referred to as “Walk Through” mutagenesis (see, e.g., U.S. Pat. Nos. 5,830,650; 5,798,208) has been used to mutagenize a defined region of a polypeptide by synthesizing a mixture of degenerate oligonucleotides that, statistically, contain a desired set of mutations. However, because degenerate polynucleotide synthesis is employed, Walk-Through mutagenesis yields a number of undesired alterations in addition to the desired set of mutations. For example, to sequentially introduce a mutation across a defined region of only five amino acid positions, a set of over 100 polynucleotide must be made (and screened) (see, e.g., FIG. 6). Accordingly, to make and screen, for example, two or three regions becomes increasingly complex, i.e., requiring the making and screening of 200 to over 300 polynucleotides, respectively, for the presence of only 10 to 15 mutations.
In yet another approach which has been used to mutagenize proteins is alanine scanning mutagenesis, where an alanine residue is “scanned” through a portion of a protein to identify positions where the protein's function is interrupted. However, this approach only looks at loss of protein function by way of substituting a neutral alanine residue at a given position, rather than gain or improvement of function. Thus, it is not a useful approach for generating proteins having improved structure and function.
Accordingly, a need remains for a systematic way to mutagenize a protein for new or improved function.